Hyperisenin A (1) was obtained as a colorless oil, and the molecular formula was defined as C30H40O4 based on its HRESIMS data (m/z [M + Na]+: 487.2802, calcd 487.2819), which possessed eleven degrees of unsaturation. The 1H NMR data showed five aromatic protons [δH 8.01 (2H, dd, J = 8.5, 1.4 Hz), 7.47 (1H, tt, J = 7.3, 1.4 Hz), 7.41 (2H, tt, J = 7.4, 1.4 Hz)], four olefinic protons [δH 6.15 (1H, d, J = 15.5 Hz), 5.98 (1H, dd, J = 15.5, 7.7 Hz), 5.15 (1H, m), 5.13 (1H, m)], and seven methyls [δH 1.74 (3H, s), 1.71 (3H, s), 1.66 (3H, s), 1.60 (3H, s), 1.38 (3H, s), 1.37 (3H, s), 1.07 (3H, s)] (Table 1). The 13C NMR spectrum showed 30 carbon signals (Table 1), which could be assigned to one ketone carbonyl (δC 196.0), one monosubstituted phenyl ring [δC 132.0, 130.5 (× 2), 129.4, 127.8 (× 2)], eight olefinic carbons (δC 166.8, 144.8, 135.6, 133.4, 122.3, 121.9, 119.0, 117.6), and three quaternary carbons including two oxygenated carbons (δC 76.3, 71.0), two methines, three methylenes and seven methyls. By analyzing the 1D NMR data and unsaturation, combined with the data reported in the literature [28,29,30], it is inferred that 1 was a polyprenylated acylphloroglucinol with a bicyclic system.

Its gross structure was confirmed by analyzing its 1D and 2D NMR data (Fig. 2). The HMBC correlations from H2-4 to C-2, C-3, and C-6, from H3-25 to C-1, C-5, and C-6, from H-13 to C-7, the 1H-1H COSY cross-peaks of H2-4/H-5 and H-9/H-10/H-11/H-12/H-13, combined with chemical shifts of C-1 (δc 117.6) and C-7 (δc 166.8), indicated that the cyclohexanone system existed, and a benzoyl group converted into enol form was attached at C-1. Then, the HMBC correlations from H2-15 to C-2, C-3, and C-4, from H3-18 to C-16, C-17, and C-19, from H3-24 to C-21, C-22, and C-23, from H3-25 to C-6, from H3-30 to C-28 and C-29, combined with the 1H-1H COSY cross-peaks of H2-15/H-16, H-5/H2-20/H-21, and H-26/H-27/H-28, revealed the existence of three side chains at C-3, C-5, and C-6. Further analysis of the downfield chemical shifts of C-3 (δc 76.3) and C-26 (δc 94.0) indicated that hydroxyl groups were attached to C-3 and C-26, respectively. The above-mentioned groups accounted for ten degrees of unsaturation. The remaining degree of unsaturation was attributed to a furan ring formed between C-7 and C-26, which was consistent with both the downfield chemical shift of C-26 (δc 94.0) and the HRESIMS data. Accordingly, the planar structure, featuring a cyclohexanone-monocyclic skeleton, was confirmed.

Key 2D NMR correlations of compounds 1–4

The relative configurations of C-5, C-6, and C-26 in 1 were assigned by the NOESY spectrum, in which the cross-peaks of H-5/H-26 and H3-25/H-27 indicated that H3-25 was α-oriented, while H-5 and H-26 were on the same side with the β-orientations. The large coupling constant between H-27 and H-28 (J = 15.5 Hz) confirmed the E configuration. In order to determine the relative configuration of C-3, the calculated 13C NMR data with DP4+ analysis of two configurations (3R*,5R*,6S*,26R*)-1 and (3S*,5R*,6S*,26R*)-1 were applied at the mPW1PW91/6–311 + G** level. The results showed that (3R*,5R*,6S*,26R*)-1 had a better linear correlation with 100% DP4+ probability (R2 = 0.9990) (Fig. 3A), suggesting that the relative configuration of 1 was defined as 3R*,5R*,6S*,26R*. The absolute configuration of 1 was identified by ECD calculation at the PBE0/def2-TZVP level, the result showed that the calculated ECD (3R,5R,6S,26R)-1 curve matched well with the experimental one, allowing to assign its absolute configuration as 3R,5R,6S,26R (Fig. 4). Thus, the structure of 1 was elucidated.

(A) NMR calculations with a DP4+ probability analysis: (3R*,5R*,6S*,26R*)-1 and (3S*,5R*,6S*,26R*)-1. (B) Linear correlation between the experimental and calculated 1H (left) and 13C NMR (right) chemical shifts, and the results of a DP4+ probability analysis for (1R*,3R*,5S*,6S*,16R*)-2

Experimental and calculated ECD spectra of compounds 1–4

Hyperisenin B (2), a colorless oil, possesses the molecular formula C31H40O5 on the basis of the HRESIMS data (m/z [M + Na]+: 515.2753, calcd 515.2768). Its 1H and 13C NMR spectra showed the presence of a monosubstituted benzene ring, two ketone carbonyls, one ester carbonyl, six olefin carbons, and seven methyl groups, which were similar to those of spirohypolactone B [31]. Further comparison of NMR data between 2 and spirohypolactone B revealed that they possessed the same skeleton, except for the acyl substituent and the number of the double bond [31]. The presence of a phenyl group at C-7 was verified by the 1H-1H COSY cross-peaks of H-9/H-10/H-11/H-12/H-13 and the HMBC cross-peak from H-13 to C-7, which was consistent with its 1D NMR data (Fig. 2). Furthermore, the 4-methylpenta-1,3-diene group was attached at C-6, deduced from the HMBC correlations from H3-25 to C-6 and C-26, from H3-30 to C-31, from H3-31 to C-28, and the 1H-1H COSY cross-peaks of H-26/H-27/H-28. The relative configurations was the same as that of spirohypolactone B and norhyperpalum H assigned as 1R*,3R*,5S*,6S*,16R* via the similar the NOESY cross-peaks of H-1/H-5, H2-20/H3-25, H2-4a/H2-15b, and H2-4b/H3-25, and along with the crucial absence of the NOESY cross-peaks of H2-4/H-16 and H-5/H2-15 (Fig. 2). To further confirm the relative configurations of C-3 and C-16, a DP4+ probability analysis of four isomers [A: (3R*,16R*), B: (3R*,16S*), C: (3S*,16R*), D: (3S*,16S*)] was conducted, and the calculated data of (1R*,3R*,5S*,6S*,16R*)-2 has good linear correlations with the experimental data with 100% DP4+ probability (all data) (Fig. 3B and S40). This deduction was further supported by a critical 1D NMR comparison in the same solvent of 2 with similar compounds, spirohypolactones A and B, and norhyperpalum H [31, 32] (Figure S41). The Δ26(27) double bond was assigned as E configuration based on the coupling constant (JH-26, H-27 = 15.4 Hz). Finally, the ECD calculation of 2 was conducted at the PBE0/def2-TZVP level, and its absolute configuration was determined to be 1R,3R,5S,6S,16R (Fig. 4).

Hyperisenin C (3), obtained as a yellow oil, had the molecular formula C38H50O5 based on its HRESIMS data (m/z [M + Na]+: 609.3572, calcd 609.3550), suggesting fourteen indices of hydrogen deficiency. Its 1H NMR spectrum showed the existence of five characteristic protons of the benzene ring [δH 7.57 (2H, dd, J = 8.2, 1.1 Hz), 7.52 (1H, tt, J = 7.5, 1.3 Hz), 7.37 (2H, t, J = 7.8 Hz)], three olefinic protons [δH 5.56, (1H, m), 5.22 (1H, m), 4.52 (1H, m)], and nine methyl groups [δH 1.81 (3H, s), 1.76 (3H, s), 1.70 (3H, s), 1.68 (3H, s), 1.59 (3H, s), 1.48 (3H, s), 0.96 (3H, s), 0.81 (3H, s), 0.76 (3H, s)] (Table 1). Its 13C NMR and DEPT data indicated 38 carbons (Table 1). The 1D NMR spectra characteristics of the above analysis indicated that 3 belonged to a polyprenylated acylphloroglucinol derivative.

Detailed analysis of its HSQC, HMBC, and 1H–1H COSY spectra indicated that 3 had the same skeleton as that of madeleinol A [33], and the main differences were acyl side chain and isopentenyl side chain (Fig. 2). The HMBC correlations from H2-14 to C-2, C-3, and C-4, from H3-17 to C-15 and C-18, from H3-32 to C-30, C-31, and C-33, from H3-33 to C-27, from H3-37 to C-35 and C-38, combined with the 1H–1H COSY correlations of H-27/H2-34/H-35 and H2-14/H-15, suggested the location of the gem-dimethyl group, and two isoprenyl groups located at C-3 and C-27, respectively. Furthermore, the O-isoprenyl side chain was attached at C-4, which was deduced from the HMBC correlations from H2-19 to C-4, from H3-23 to C-20 and C-22, along with the 1H–1H COSY cross-peaks of H2-19/H-20, and the downfield chemical shift of C-19 (δC 71.1). In addition, the benzene ring was attached at C-7 by analysis of its 1D NMR data and the HMBC correlation from H-9 to C-7, and the 1H–1H COSY cross-peaks of H-9/10/H-11/H-12/H-13. In the NOESY spectrum (Fig. 2), the cross-peaks of H-25/H-30, H-27/H-30, and H-30/H3-33 indicated that these groups were cofacial, assigned as α-orientations, while H-24β/H3-28, H3-28/H-29β, and H-29β/H3-32 clarified that they were β-oriented. The calculated ECD method was applied to determine its absolute configuration as 25R,26S,27S,30S (Fig. 4). Thus, a unique O-prenylated acylphloroglucinol with a 6/6/6 ring system was established.

Hyperisenin D (4) was also obtained as a yellow oil and had the molecular formula C33H42O6 based on its HRESIMS at m/z 557.2876 [M + Na]+ (calcd 557.2874), which possessed thirteen degrees of unsaturation. The 1D NMR and HSQC spectra revealed 33 carbons (Table 1), including a benzoyl group [δc 193.9, 138.0, 133.5, 129.6 (× 2), 128.5 (× 2)], a geranyl group (δc 141.4, 132.1, 123.8, 116.7, 40.1, 36.3, 26.8, 25.9, 17.9, 16.4), two oxidated quaternary carbon (δc 72.2, 71.2), two methines (δc 93.0, 90.8), two methylenes (δc 31.6, 28.0), and four methyl groups (δc 27.6, 25.5, 23.8, 23.4), and the remaining six carbons were characteristic of a dearomatized phloroglucinol core including an enolic 1,3-diketone moiety (δc 182.3, 175.7, 114.7), one oxygen-bearing ene (δc 170.0, 113.3), and one quaternary carbon (δc 50.5). The mentioned groups occupied eleven degrees of unsaturation. Two additional rings should be formed in the structure of 4. Thus, the aforementioned evidence suggested that 4 should be a tricyclic dearomatized prenylated acylphloroglucinol derivative.

The planar structure was confirmed by analyzing its HSQC, HMBC, and 1H-1H COSY spectra (Fig. 2), similar to that of hypermonin C (5) [34], and the main difference was the isoprenyl side chain at C-5. The HMBC correlations from H2-19 to C-5, C-6, and C-24, from H3-22 to C-20, from H3-23 to C-20 and C-21, combined with the 1H-1H COSY cross-peaks of H2-19/H-20, indicated that the oxidized isopentenyl side chain was located at C-5. Furthermore, the downfield chemical shift of C-20 (δC 90.8), combined with degrees of unsaturation, indicated that C-6 and C-20 were connected via an oxygen atom to form a furan ring. In the NOESY spectrum (Fig. 2), the cross-peaks of H3-18/H3-27 and H-20/H2-24 indicated that H-20 and the geranyl group at C-5 were on the same side, assigned as β-orientations, while H-15 was on the opposite side with α-orientation. Then, the NOESY cross-peaks of H-25/H2-28 revealed the E-configuration of C-25/C-26 double bond. Thus, the relative configuration of 4 was determined to be 5S*,15S*,20R*. Finally, the absolute configuration of 4 was defined as 5S,15S,20R by ECD calculation (Fig. 4).

Two known compounds, hypermonin C (5) [34] and vismiaguianone B (6) [35], were obtained from this plant. Their structures were confirmed by comparing the 1D NMR data with those of the literature.

Hyperisenins A (1) and B (2), possessing a unique cyclohexanone-monocyclic system, were proposed to biogenetically originate from BPAPs (Fig. 5) [28, 36, 37]. It underwent a retro-Claisen reaction to yield the crucial intermediate i, followed by two distinct pathways to form intermediates ii and iii. Subsequently, compound 1 was constructed from ii via oxidation, keto − enol tautomerism, and intramolecular cyclization. On the other hand, iii underwent oxidation and intramolecular cyclization to obtain compound 2.

Plausible biogenetic pathway of compounds 1 and 2

Considering QS is the vital target for antimicrobial therapy [13, 14] and Pseudomonas aeruginosa is an opportunistic pathogen that is typically resistant to multiple clinically available antibiotics [38], compounds 1–6 were evaluated for the QS inhibitory activity against P. aeruginosa (Figure S46). The results showed that compound 4 was a potential QS inhibitor that decreased the activation of the rhl system, as evidenced by the reduced fluorescence density of the reporter strain PAO1-rhlA-gfp (Fig. 6A). As expected, compound 4 did not affect the growth of P. aeruginosa, consistent with its role as a QS inhibitor. We further examined the production of rhamnolipids, a virulence factor regulated by the rhl system, in a clinically isolated carbapenem-resistant P. aeruginosa (CRPA) strain. Compound 4 significantly reduced rhamnolipid levels at a concentration of 100 µM (Fig. 6B). P. aeruginosa's QS system includes two other well-defined pathways apart from the rhl system: the las and pqs systems [8]. Due to the limited yield of compound 4, its potential mechanism was explored through molecular docking with three QS receptors (lasR, rhlR, and pqsR).

Compound 4 as a quorum sensing inhibitor against P. aeruginosa. A Compound 4 inhibited the activation of the rhl pathway without affecting bacterial growth. B Compound 4 reduced the expression of the virulence factor rhamnolipid in CRPA. C Docking results of compound 4 with lasR (PDB 6D6L, colored by chain), depicted as slate sticks. D Docking results of compound 4 with pqsR (PDB 6B8A, colored by chain), shown as slate sticks. E Proposed mechanism of inhibition by compound 4 against rhamnolipid production, potentially through competitive inhibition of the lasR and pqsR receptors, both of which enhance the activation of the rhl system and rhamnolipid production. Data are presented as mean ± SD (n = 3). Significance levels are indicated as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Unexpectedly, compound 4 failed to dock with rhlR but successfully engaged with lasR and pqsR, demonstrating significant affinity within their ligand-binding pockets. For lasR, it is proposed that compound 4 forms two aromatic hydrogen bonds with Tyr47 and a combination of a hydrogen bond and an aromatic bond with Tyr56, yielding a docking score of -6.476 kcal/mol (Fig. 6C). Regarding pqsR, compound 4 appears to bind to the ligand-binding site, albeit slightly shifted towards the dimer interface, forming a hydrogen bond with Glu151 in chain A and both a hydrogen bond and an aromatic bond with Glu151 in chain B, resulting in a docking score of -6.217 kcal/mol (Fig. 6D). Given that both the las and pqs systems enhance the activation of the rhl system [8], we hypothesize that the binding of compound 4 to lasR and pqsR may competitively inhibit their contribution to the rhl system, thereby regulating rhamnolipid production (Fig. 6E).

In summary, the phytochemical investigation of the dried aerial parts of H. seniawinii Maxim. resulted in the isolation of four undescribed polyprenylated acylphloroglucinols (1–4), as well as two known analogs (5 and 6). All isolates were obtained from this plant for the first time. Compounds 1 and 2 were two degraded polyprenylated acylphloroglucinols bearing the unique cyclohexanone-monocyclic system, and their plausible biosynthetic pathway was proposed. Furthermore, compound 4 was a potential QS inhibitor that decreased the activation of the rhl system and reduced rhamnolipid levels. Its mechanism might be the ability to bind between 4 lasR and pqsR. Our findings might provide a potential candidate as QS inhibitors to treat infectious diseases for further research.